The bioprocessing industry has traditionally used stainless steel systems and piping in manufacturing processes for fermentation and cell culture. These devices are designed to be steam sterilized and reused. Cleaning and sterilization are however costly labor-intensive operations. Moreover, the installed cost of these traditional systems with the requisite piping and utilities is often prohibitive. Furthermore, these systems are typically designed for a specific process, and cannot be easily reconfigured for new applications. These limitations have led to adoption of a new approach over the last ten years—that of using plastic, single-use disposable bags and tubing, to replace the usual stainless steel tanks.
In particular bioreactors, traditionally made of stainless steel, have been replaced in many applications by disposable bags, which are rocked to provide the necessary aeration and mixing necessary for cell culture. These single-use bags are typically provided sterile and eliminate the costly and time-consuming steps of cleaning and sterilization. The bags are designed to maintain a sterile environment during operation thereby minimizing the risk of contamination.
Commonly used bags are of the “pillow style,” mainly because these can be manufactured at low cost by seaming together two flexible sheets of plastic.
One of the successful disposable bioreactor systems uses a rocking table on to which a bioreactor bag is placed. The bioreactor bag is partially filled with liquid nutrient media and the desired cells. The table rocks the bag providing constant movement of the cells in the bag and also aeration from the turbulent air-liquid surface. The bag, typically, has a gas supply tube for the introduction of air or oxygen, and an exhaust gas tube to allow for the removal of respired gases. Nutrients can be added through other tubes.
One possible limitation of this type of device is that it may be difficult to scale up beyond a few hundred liters because poor liquid circulation causes nutrient and waste gradients that inhibit cell performance. This is because the back and forth motion of the single-axis rocker used in these applications creates good liquid circulation in the direction perpendicular to the rocking axis, but relatively little mixing in the direction parallel to the rocking axis. In large volume bags (greater than 100 liters), or in bags with a large length to width ratio, this poor axial circulation can result in a long time to achieve homogeneity of the bag contents. This makes pH control in the bioreactor bag difficult, since additions of acid or base added to the bioreactor to modulate the pH can take a long time to disperse throughout the bag. Nutrients added to the bioreactor bag may not be distributed uniformly. Poor liquid circulation also limits the amount of oxygen that can be transferred from the headspace, and thus the maximum concentration of cells that cannot be cultured.
Circulation flow can be improved by incorporating a second axis of rotation. By synchronizing the two axes it is possible to impart a gyratory motion that greatly improves mixing and mass transfer. However, the addition of second axis increases the cost tremendously, and the increase in mechanical complexity makes the rocker less reliable and more difficult to maintain.
However, all of the above limitations are present in the U.S. Pat. No. 6,190,913 to Vijay Singh (and the resultant Wave Bioreactor marketed by GE Healthcare); as a result, the same inventor proposed in the U.S. patent application Ser. No. 12/676,180 (assigned to GE Healthcare) filed Sep. 15, 2008 to overcome these limitations by creating flexible baffles inside the bag to create a swirling motion in the bag to improve mixing. However, this modification of the existing art does not remove the most significant drawback of producing stress on the seams of the bag that prevent the use of large-scale production using these bags. The proposed modification also does not provide complete mixing as desired from all directions in the bag since the bag is rocked only in one dimension.
Therefore, there is a need for an apparatus that enables a user to scale up the mixing of nutrient media in a bioreactor bag in a flexible container that will assure complete mixing nutrient media from all directions.